Sample Block Apparatus and Method for Maintaining a Microcard on a Sample Block

ABSTRACT

A thermal cycling device for thermally cycling samples of biological material contained in a microcard having a top and bottom surface. The thermal cycling device can include a sample block having an upper surface configured for engaging the bottom surface of a microcard, a vacuum device, and a temperature control system operatively connected with the sample block. The upper surface of the sample block may include a plurality of channels, the channels defining spaces between the sample block and the bottom surface of a microcard that may be positioned thereon. The vacuum device may be in fluid communication with the sample block for drawing gas out of the spaces defined by the channels in the sample block. The vacuum device may be configured for substantially maintaining a vacuum between the sample block and microcard so that a retention force is imparted on the microcard to urge the microcard toward the sample block. Methods of maintaining a microcard on a sample block of a thermal cycling device are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/207,263 filed on Jul. 30, 2002. The entire disclosure of the aboveapplication is incorporated herein by reference.

FIELD

The present teachings relate generally to sample block apparatussuitable for use in a thermal cycling device, and methods of maintaininga microcard on a sample block of a thermal cycling device. Moreparticularly, the present teachings further relate, in various aspects,to sample block apparatus utilizing a vacuum to maintain a microcard ona sample block during a nucleic acid amplification process such aspolymerase chain reaction (PCR).

BACKGROUND

Biological testing has become an important tool in detecting andmonitoring diseases. In the biological testing field, thermal cycling isused to amplify nucleic acids by, for example, performing PCR and otherreactions. PCR in particular has become a valuable research tool withapplications such as cloning, analysis of genetic expression, DNAsequencing, and drug discovery.

Recent developments in the field have spurred growth in the number oftests that are performed. One method for increasing the informationobtainable through such biological testing is to provide real-timedetection capability during thermal cycling. During real-time detectionthe characteristics of samples of biological materials can be detectedwhile the sample well tray remains positioned in the thermal cyclingdevice. A method for increasing throughput is to place a large number ofsamples on a single microcard. In this manner, more tests may beperformed in a given period of time. Moreover, it is possible to reducecosts by running at low reaction volumes of biological materials. It mayalso be desirable for there to be substantial temperature uniformitybetween the plurality of samples on a single microcard.

SUMMARY

Various aspects generally relate to, among other things, a thermalcycling device for thermal cycling samples of biological materialcontained in a microcard.

Various aspects provide a thermal cycling device for thermally cyclingsamples of biological material contained in a microcard having a top andbottom surface. The thermal cycling device can comprise a sample blockhaving an upper surface configured for engaging the bottom surface of amicrocard, a vacuum device, and a temperature control system operativelyconnected with the sample block. The upper surface of the sample blockmay include a plurality of channels, the channels defining spacesbetween the sample block and the bottom surface of a microcard that maybe positioned thereon. The vacuum device may be in fluid communicationwith the sample block for drawing gas out of the spaces defined by thechannels in the sample block. The vacuum device may be configured forsubstantially maintaining a vacuum between the sample block andmicrocard so that a retention force is imparted on the microcard to urgethe microcard toward the sample block. The temperature control systemmay be configured for cycling the sample block through a sequence oftimes and temperatures comprising at least a first temperaturemaintained for a first period of time and a second temperaturemaintained for a second period of time, with the second temperaturebeing higher than the first temperature, and the cycling comprising atleast two repetitions of said sequence of time and temperatures.

Various other aspects comprise a sample block apparatus for a thermalcycler configured for use with a microcard containing a plurality ofsamples of biological material. The sample block apparatus can comprisea sample block, a vacuum source, and a temperature control systemoperatively connected with the sample block to cycle the sample blockaccording to a user-defined profile. The sample block can comprise anupper surface configured for resting a microcard thereon, the uppersurface including surface irregularities for defining spaces between thesurface irregularities and a microcard that may be positioned thereon.The vacuum source may be in fluid communication with the space betweenthe surface irregularity and the microcard positioned thereon. Thevacuum source may be configured to create a substantial vacuum in thespaces thereby imparting a force on the microcard to retain themicrocard on the sample block upper surface.

Further various aspects comprise a microcard retaining apparatus for athermal cycler of biological materials. The microcard retainingapparatus can comprise a sample block having a an upper surface and avacuum port. The sample block upper surface may be substantially flatand configured to engage a bottom surface of a microcard that may bepositioned thereon. The upper surface of the sample block may furthercomprise a plurality of recesses. The vacuum port in the sample blockmay be in fluid communication with the plurality of recesses to assistin imparting a vacuum in the recesses to cause the microcard to pressdownward against the upper surface of the sample block. The vacuum portmay be configured for attachment to a vacuum source.

Various aspects also comprise a method of maintaining a microcard on asample block of a thermal cycling device. The method can include thesteps of providing a sample block with a plurality of channels on anupper surface thereof. The method may further include the step ofproviding a space for a microcard containing at least one sample ofbiological material above the upper surface of the sample block so thata bottom surface of the microcard may contact the upper surface of thesample block. A vacuum may be imparted on the spaces defined by thechannels on the upper surface of the sample block and the bottom surfaceof the microcard positioned adjacent the upper surface of the sampleblock, the vacuum creating a force to urge the microcard against theupper surface of the sample block. The microcard may then be thermallycycled through a sequence of times and temperatures comprising at leasta first temperature maintained for a first period of time and a secondtemperature maintained for a second period of time, with said secondtemperature being higher than said first temperature. Optionally,simultaneously with the step of thermally cycling the microcard, theoptical characteristics of the at least one sample of biologicalmaterial may be detected.

Further various aspects comprise an apparatus for thermally cyclingsamples of biological material contained in a microcard. The apparatuscan comprise a sample block configured to assist in heating and coolinga microcard during thermal cycling, means for urging a microcard againsta top surface of a sample block of a thermal cycling device using avacuum, and means for imposing a substantial vacuum in a space betweenthe sample block and the microcard.

Still further various aspects comprise a thermal cycling apparatus. Thethermal cycling apparatus can comprise a base, with said base defining avoid therein, a vacuum port disposed for fluid communication with thevoid, support features disposed adjacent the void on the base, and atemperature control system. The support features include uppermostsurface regions defining a common plane. The temperature control systemmay be configured for cycling at least one of the base and the supportfeatures through a sequence of times and temperatures comprising atleast a first temperature maintained for a first period of time and asecond temperature maintained for a second period of time, with saidsecond temperature being higher than said first temperature and saidcycling comprising at least two repetitions of said sequence of timesand temperatures.

It is to be understood that both the foregoing general description andthe following description of various embodiments are exemplary andexplanatory only and are not restrictive.

DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several exemplary embodiments. Inthe drawings,

FIG. 1 is a top perspective view of an exemplary embodiment of a thermalcycling device according to the present teachings;

FIG. 2 is bottom perspective view of a microcard and microcard carrierof the thermal cycling device shown in FIG. 1;

FIG. 3 is a side cross-sectional view of the thermal cycling device ofFIG. 1 along line III-III of FIG. 1, in an assembled state;

FIG. 4 is a close up cross-sectional view of a portion of the thermalcycling device of FIG. 3;

FIG. 5 is a top perspective view of a thermal cycling device accordingto another embodiment of the present teachings;

FIG. 6 is a top perspective view of a sample block platform, microcard,and microcard carrier according to another embodiment of the presentteachings;

FIG. 7 is a bottom perspective view of the sample block platform,microcard, and microcard carrier of FIG. 6;

FIG. 8 is a cross-sectional view of the sample block platform,microcard, and microcard carrier along line VIII-VIII of FIG. 6, in anassembled state; and

FIG. 9 is a close up cross-sectional view of a portion of the sampleblock platform, microcard, and microcard carrier of FIG. 8.

DETAILED DESCRIPTION

Reference will now be made to various exemplary embodiments, examples ofwhich are illustrated in the accompanying drawings. Wherever possible,the same reference numbers are used in the drawings and the descriptionto refer to the same or like parts.

In accordance with various embodiments, a thermal cycling device isprovided. In various aspects, the thermal cycling device may performnucleic acid amplification on a plurality of biological samplespositioned in a microcard. In various embodiments, the thermal cyclingdevice includes a sample block. In various embodiments, the thermalcycling device may also include a microcard carrier and a cover. Variousembodiments are directed toward a sample block apparatus comprising asample block, a vacuum source, and a temperature control system.

Although terms like “horizontal,” “vertical,” “upward,” and “downward”may be used in describing various aspects of the present teachings, itshould be understood that such terms are for purposes of more easilydescribing the teachings, and do not limit the scope of the teachings.

In various embodiments, such as illustrated in FIGS. 1-4, the thermalcycling device 10 configured for use with a microcard 12 includes asample block apparatus 30, a microcard carrier 70, and a cover 80. Thethermal cycling device may be configured to perform nucleic acidamplification on the samples of biological material. One common methodof performing nucleic acid amplification of biological samples ispolymerase chain reaction (PCR). Various PCR methods are known in theart, as described in, for example, U.S. Pat. Nos. 5,928,907 and6,015,674 to Woudenberg et al., the complete disclosures of which arehereby incorporated by reference for any purpose. Other methods ofnucleic acid amplification include, for example, ligase chain reaction,oligonucleotide ligations assay, and hybridization assay. These andother methods are described in greater detail in U.S. Pat. Nos.5,928,907 and 6,015,674.

In various embodiments, the thermal cycling device performs real-timedetection of the nucleic acid amplification of the samples in themicrocard during thermal cycling. Real-time optical detection systemsare known in the art, as also described in greater detail in, forexample, U.S. Pat. Nos. 5,928,907 and 6,015,674 to Woudenberg et al.,incorporated herein above. During real-time detection, variouscharacteristics of the samples are detected during the thermal cyclingin a manner known in the art. Real-time detection permits more accurateand efficient detection and monitoring of the samples during the nucleicacid amplification. In the embodiment shown in FIGS. 1-4, an opticaldetection system (not shown) is positioned above the microcard 12.

The thermal cycling device 10 shown in FIGS. 1-4 is particularly suitedfor use with a microcard. The microcard may be, in various embodiments,any type of two-dimensional array of sample loci held within acontinuous or non-perforated substrate. This substrate may be flexibleor rigid. The substrate or microcard may include any number of samplechambers for containing samples of the biological material. The mosttypical number of sample chambers is 60, 96, 384, or 1536, however, themicrocard may include any other number of sample chambers from one to atleast several thousand. FIG. 1 shows an example of a microcard sampletray having 384 wells. Several non-limiting examples of some sample welltrays of the microcard type suitable for use in the present inventionare described in WO 02/01180 to Bedingham et al., the completedisclosure of which is hereby incorporated by reference for any purpose,WO 01/28684 to Frye et al., the complete disclosure of which is herebyincorporated by reference for any purpose, and WO97/36681 to Woudenberget al., the complete disclosure of which is hereby incorporated byreference for any purpose. Any number of other types of microcards arealso contemplated for use herein.

As embodied herein and shown in FIGS. 1-4, microcard 12 is rectangularin shape. It should be understood that the microcard may be any othersuitable shape. The microcard 12 has a top surface 14 and a bottomsurface 16. The microcard may be made out of one or several pieces. Inthe example shown in FIGS. 1-4, the sample well tray includes 384 samplechambers 18 positioned in a well-known 16.times.24 array. The samplechambers may be loaded with biological materials in any of a variety ofknown manners (e.g., micropipetting). The volume of the sample chambersmay vary depending on the number of sample chambers, and the specificapplication.

In the embodiment shown in FIG. 1, the microcard may include a pair ofnotches 20 for engaging with a microcard carrier in a manner that willbe described below. It is contemplated that the microcard may beprovided without such notches however.

In accordance with various embodiments, the thermal cycling deviceincludes a sample block apparatus configured to receive the microcardthereon. As described herein and shown in FIGS. 1-4, the sample blockapparatus is generally designated by the reference number 30. It is tobe understood that the sample block apparatus shown in FIG. 1-4 is byway of example only, and the present teachings are not limited to thesample block apparatus shown in FIGS. 1-4. In the embodiment shown inFIGS. 1-4, sample block apparatus (or sample block) 30 comprises asample block base 32 and sample block platform 34. Sample block platformis positioned on an inner region of the sample block base 32. A groove40 on the top surface 36 of the sample block base 32 defines a recess inwhich the sample block platform 34 may be positioned.

Sample block platform 34 comprises a raised upper region with a topsurface 52, and a support 37. The support 37 includes a flat uppersurface 38 and angled support member 39. In the embodiment shown, thesample block platform may be removably attached to the sample block base32 via a fastening member 42. In the example shown, the fastening member42 is a threaded fastener. Any other type of fastening member may alsobe suitable. In other embodiments, the sample block platform may beintegral with the sample block base 32.

The sample block base 32 and sample block platform 34 may be made out ofany suitable material, such as aluminum, gold-plated silver, or athermally-conductive polymer/plastic. The material can be heatconductive so that the sample block may assist in thermal cycling. Thesample block base 32 may be attached to any known type of heat sink. Inthe embodiment shown in FIGS. 1-4, the heat sink is a finned heat sink44. The sample block typically includes at least one heating element. Invarious embodiments, the at least one heating element includes a peltierheater. Other types of heating elements may be used instead of, or incombination with, the peltier heater. A convection unit such as a fanmay also be positioned adjacent the heat sink 44.

The sample block may be operatively connected to a temperature controlsystem programmed to raise and lower the temperature of the sample blockaccording to a user-defined profile. Several non-limiting examples ofsuitable temperature control systems for raising and lowering thetemperature of the sample block are described in U.S. Pat. No. 5,656,493to Mullis et al. and U.S. Pat. No. 5,475,610 to Atwood et al., thedisclosures of which are both hereby incorporated by reference for anypurpose. For example, in various embodiments, a user supplies datadefining time and temperature parameters of the desired PCR protocol toa control computer that causes a central processing unit (CPU) of thetemperature control system to control thermal cycling of the sampleblock. In a typical thermal cycler of the present teachings, thetemperature control system may be configured for cycling the sampleblock through a sequence of times and temperatures comprising at least afirst temperature maintained for a first period of time and a secondtemperature maintained for a second period of time, with the secondtemperature being higher than the first temperature, and the cyclingcomprising at least two repetitions of said sequence of time andtemperatures.

In accordance with various embodiments, the sample block comprises anupper surface configured for resting a microcard thereon during thermalcycling of the microcard. The upper surface includes surfaceirregularities for defining a space between selected regions of theupper surface and a microcard positioned thereon during thermal cycling.In various embodiments, the surface irregularities comprise channels orrecesses. As embodied herein, and shown in FIGS. 1-4, the sample blockplatform 34 may be a rectangular block of material with a side surface50 and an upper surface 52. As shown in FIGS. 1-5, the upper surfaceincludes a plurality of channels (or recesses or voids) positioned in aperpendicularly intersecting manner. For sake of simplifying thedescription of the channels (or recesses or voids) in the specification,the channels shown in FIGS. 1-4 will be referred to as lateral channels54 and longitudinal channels 56. As shown in FIG. 1, the lateralchannels 54 have a shorter length than the longitudinal channels 56. Inthe embodiment shown in FIG. 1, the upper surface includes seven lateralchannels 54 and nine longitudinal channels 56. Any other number oflateral and longitudinal channels may be used instead.

Although FIGS. 1-4 show the channels being positioned in aperpendicularly intersecting manner, it should be understood that thechannels may be in any other geometric shape. The perpendicularlyintersecting pattern is shown for purposes of example only. Thepositioning on the channels may be a function of the microcard features.In various embodiments, it may be generally desirable to position thechannels so that the channels are not positioned immediately below thesample chambers 18. Instead, it may be desirable to have the top flatsurface 52 contacting the bottom surface 16 of the microcard 12 in thearea immediately below the sample chambers 18. One reason for this isthat it may be desirable that the bottom surface of the microcarddirectly below the sample chamber be directly in contact with the uppersurface 52 of the sample block platform, in order to minimizetemperature differences between adjacent samples.

FIGS. 3 and 4 show the thermal cycling device of FIG. 1 in the assembledstate. The cross-section shown in FIGS. 3 and 4 is taken along a lateralchannel 54 as indicated by the line III-III in FIG. 1. FIGS. 3 and 4show the space created by lateral channel 54 under bottom surface 16 ofmicrocard 12, when the microcard 12 is placed on the upper surface 52 ofthe sample block platform. FIG. 4 also shows the width and depth oflongitudinal channel 56 that intersects with the lateral channel 54along which the cross-section is taken. When the microcard is placed onthe upper surface 52 of the sample block platform, the channels definespaces in which a vacuum may be imparted as described below. Inaccordance with the embodiment shown in FIGS. 1-4, the spaces are influid communication with one another so that a vacuum may be drawn inthe spaces.

The channels (or recesses or voids) may be formed in the upper surface52 by any known manner. The width and depth of the channels may bevaried from that shown in FIGS. 3 and 4. In FIG. 4, reference number 58indicates the lower surface of lateral channel 54. When the microcard 12is inserted into the thermal cycling device, the bottom surface 16 ofthe microcard rests flush against the upper surface 52 of the sampleblock platform. Because there remains a large amount of surface area ofthe microcard that is in contact with the upper surface 52 of the sampleblock, the sample block may effectively perform its function oftransferring heat to and away from the microcard before, during, and/orafter thermal cycling.

In various embodiments, the sample block apparatus further includes avacuum device in fluid communication with the sample block platform fordrawing gas, such as air, out of the spaces defined by the channels inthe sample block platform. The vacuum device is configured forsubstantially maintaining a vacuum between the sample block platform andmicrocard so that a downward force is imparted on the microcard to urgethe microcard toward the sample block platform. The vacuum device may beconnected to the sample block via a vacuum port positioned on or in thesample block platform. In the embodiment of FIGS. 1-4, the vacuum portis positioned along the rear edge of the sample platform, and istherefore out of view in FIGS. 1-4. The vacuum port may be an aperturewith a passage in direct communication with channels 54 and 56. Byattaching a vacuum device to the sample block platform, a vacuum may bedrawn in the channels, thereby pulling downward on the microcard. Theresulting downward force may be sufficient to retain the microcard onthe sample block platform. The downward force from the vacuum in thespaces beneath the microcard can assist in maintaining the microcardfirmly pressed against the upper surface 52 of the sample blockplatform, thereby promoting substantial temperature uniformity betweenthe sample chambers 18, if desired.

Moreover, in real-time detection apparatuses, it may be desirable tominimize the amount of structure located between the microcard and theoptical detection system. In various embodiments, the provision of thevacuum in the spaces under the microcard eliminates the need for anapparatus such as a plate positioned above the microcard that pressesagainst the upper surface in the spaces between the sample chambers. Byeliminating the need for such a pressing plate, it may be possible toutilize a greater portion of the upper surface for sample chambers. If apressing plate is not used, space does not need to be reserved forpressing on the upper surface of the microcard. It may be desirable thatthere is a sufficient initial downward force on the microcard so that aninitial vacuum can be drawn.

It should be understood that the channels 54 and 56 shown in FIGS. 1-4are not the only type of surface irregularity suitable with the presentinvention. In other embodiments of the present invention, the uppersurface 52 of the sample block platform may include a rough surfaceinstead of the channels shown in FIGS. 1-4. The rough surface ispreferably of sufficient roughness so that the spaces created by thevalleys of the irregularities can have a substantial vacuum imparted onthem. The depth of the surface irregularities may depend on the rigidityof the microcard. For example, if the microcard is very stiff it ispossible to draw a sufficient vacuum with only very smallirregularities. Other types of surface irregularities can also be usedwith the present invention.

In various embodiments, the sample block platform may further include agroove positioned around the outer periphery of the sample blockplatform channels. In the exemplary embodiment shown in FIGS. 1-4, agroove 62 is formed around the outer periphery of the upper surface 42of the sample block platform, in an area outside of the channels 54 and56. The groove surrounds the recessed area of the upper surface 42 ofthe sample block platform. In the embodiment shown in FIGS. 3 and 4, thegroove is rectangular in shape, however, any other suitable shape isalso acceptable. In the embodiment shown in FIG. 4, a gasket 64 isinserted in the groove 62 to surround the recessed area. The gasket maybe configured to engage a bottom surface 16 of the microcard carrier 12.The gasket may be any type or shape of gasket capable of facilitating avacuum seal between the microcard 12 and sample block platform 34. Invarious embodiments, the gasket is suitable for use in a thermal cyclingdevice. It should be understood that a complete vacuum may not benecessary. A substantial vacuum may be sufficient to initiate a downwardurging force from the microcard onto the gasket and upper surface 52 ofthe sample block platform.

In accordance with various embodiments, the thermal cycling device mayalso include a microcard carrier. As described herein and shown in FIGS.1-4, the microcard carrier 70 is a rectangular shaped object with alength and width slightly larger than the microcard. In the presentinvention, the microcard carrier serves the purpose of pressingdownwardly around the outside periphery of the top surface 14 of themicrocard 12. In the embodiment shown in FIGS. 1-4, the microcardcarrier includes a downwardly projecting rib 74 on a lower surface ofthe microcard. The microcard carrier is optional, but it can be helpfulfor use with flexible microcards.

In the embodiment shown in FIGS. 1-4, the microcard carrier 70 includesa plurality of optical openings 72 for permitting light to pass throughbetween optical detection system and the sample chambers duringdetection (e.g., real-time detection) of the biological samples in thesample chambers 18. In the embodiment shown in FIGS. 1-4, the pluralityof openings 72 are aligned with the sample chambers 18. The downwardlyprojecting rib 74 of the microcard carrier 70 may be positioned aroundthe outer periphery of the optical openings 72. In the embodiment shown,the rib 74 presses downwardly on an outer periphery of the microcard 12.The engagement of the rib with the microcard assists in sealing themicrocard 12 against gasket 64 to form an initial seal. In variousembodiments, the microcard carrier can be removed after the vacuum isdrawn below the microcard.

The microcard carrier 70 may also include a pair of guide members 76 forengagement with the notches 20 that may be provided in the microcard 12.The guide members 76 and notches 20 may assist in preventing horizontalmovement between the microcard and the microcard carrier. In variousembodiments, the microcard and microcard carrier may snap-fit together.It should be understood that the guide members and notches are optional.

In various embodiments, the thermal cycling device may also comprise acover. FIG. 1 shows a cover 80 which may be heated by heating element88. Alternately, in various embodiments, the cover might not be heated.The heated cover 80 of FIGS. 1-4 includes, among other things, a topplate 82, bottom plate 84, a plurality of spring elements 86, andheating element 88. The top plate 82 includes a plurality of opticalopenings 92 for permitting light to pass through between the opticaldetection system and the sample chambers during detection (e.g.,real-time detection) of the biological samples in the sample chambers18. The cover may assist in evenly distributing the force imparted onthe microcard by microcard carrier 70. It should be understood that thecover is optional. In the embodiment shown in FIGS. 1-4, the cover 80may assist in providing the downward force on microcard 12. In oneembodiment, the bottom plate 84 may be pushed downward, thereby pullingdown on spring elements 86, thereby pushing top plate 82 in the downwarddirection. Top plate 82 may then press downward on microcard carrier 70,which then presses downward on microcard 12 via the downwardlyprojecting rib 74.

In various embodiments, a seal can be maintained between microcard 12and sample block platform 34 without a microcard carrier and cover. Thiscan be true, for example, when the microcard carrier has a highrigidity. A rigid microcard may tend to be more resistant to warpingthan a flexible microcard, and therefore may be able to maintain a sealwith the gasket without any external force (such as a microcard carrier)pressing downward on it. If the microcard is very flexible and prone towarping, it may be helpful to provide some type of device for pressingdownward on the microcard in the area adjacent the gasket.

An operation of the thermal cycling device for the embodiment of FIGS.1-4 is described below. First, the microcard carrier 70 and a microcard12 with samples of biological material (e.g., DNA) are positioned on theflat upper surface 52 of the sample block platform. The microcardcarrier is positioned such that downwardly projecting rib 74 engages thetop portion of microcard 12. The cover 80 may then be placed over thetop of the microcard and microcard cover. In the embodiment shown inFIGS. 1-4, the cover 80 may assist in pressing downward on the microcardcarrier 70 and microcard 12. The outer periphery of microcard 12 is thusfirmly pressed against the top surface of gasket 64.

Next, a vacuum source is attached to a vacuum port on the side of thesample block so that any air positioned in the spaces defined bychannels 54 and 56 and a bottom surface 16 of the microcard isevacuated. When the space is at a substantial vacuum, the microcard willbe pulled downward by the vacuum so that the microcard is firmly pressedagainst the top flat surface 52 of the sample block platform 34. In thismanner, no large forces are needed on the top central portion of themicrocard and substantial temperature uniformity across the samplechambers may be achieved, if desired. Thermal cycling of the apparatusmay now be performed, with or without real-time detection by the opticaldetection system. During thermal cycling, the temperature control systemof the thermal cycling device is operatively connected to the sampleblock to cause the temperature of the sample block to raise and loweraccording to a pre-programmed protocol. In one embodiment, the sampleblock (and microcard) are thermally cycled through a sequence of timesand temperatures comprising at least a first temperature maintained fora first period of time and a second temperature maintained for a secondperiod of time. The second temperature is higher than the firsttemperature. The thermal cycling includes at least two repetitions ofthe sequence of time and temperatures. After the thermal cycling iscompleted, the microcard and microcard carrier may then be removed.

Further various embodiments of the thermal cycling device contemplatestructure such as shown in FIG. 5. The thermal cycling device of FIG. 5is generally designated by the reference number 110. To the extent thatthe following structure is identical to the structure described forFIGS. 1-4, a description will not be repeated. FIG. 5 shows a thermalcycling device comprising a sample block, microcard, microcard carrier,and heated cover. The microcard 112 is essentially identical tomicrocard 12 of FIGS. 1-4, however, it lacks notches for engagement witha microcard carrier. The microcard carrier 170 is similar to themicrocard carrier 70 of FIGS. 1-4. Heated cover 180 may be slightlydifferent from heated cover 80 of FIGS. 1-4. The sample block platform,however, may be identical to the sample block platform 34 describedabove for FIGS. 1-4, and is therefore also labeled with reference number34. The sample block platform 34 has the same channels on the upper flatsurface as described for FIGS. 1-4, and operates in an identical manner.Unlike FIGS. 1-4, FIG. 5 shows the outer walls of the sample block base132, as is known in the art.

The operation of the thermal cycling device for the embodiment of FIG. 5corresponds to the operation described above for the embodiment shown inFIGS. 1-4, therefore a description of the operation will not berepeated. Moreover, the same reference numbers are used to refer to thesame or like parts as shown in the embodiment of FIGS. 1 and 2A-2C. Itshould be understood that, similar to the FIGS. 1-4 embodiment, cover180 and microcard carrier 170 are optional.

FIGS. 6-8 show still further embodiments of a sample block platform,microcard, and microcard carrier according to the present invention. Inparticular, FIGS. 6-8 show a microcard having 96 chambers. An example ofa microcard of this type is described in WO 01/286684 to Frye et al.,incorporated herein above. Microcard 212 includes a top surface 214 andbottom surface 216. The microcard includes 96 chambers 218 that arefluidly connected to one another as described in greater detail in WO01/286684. In the embodiment shown in FIGS. 6-8, the microcard can befilled via sample inlet port 220 and longitudinal delivery passageways222, using, for example, a vacuum.

The embodiment of FIGS. 6-8 includes a sample block platform similar tothat shown in FIGS. 1-4 and FIG. 5, with several exceptions. The sampleblock platform 234 shown in FIGS. 6-8 includes a plurality oflongitudinal channels 256 in addition to two lateral channels 258connected to the ends of the longitudinal channels. In variousembodiments, each of the channels is fluidly connected to one another sothat a vacuum may be drawn in the space below the microcard when themicrocard is placed on the upper surface 252 of the sample blockplatform. When the microcard is placed on top of the sample blockplatform, a space is defined by the channels 256 and 258 and the bottomsurface 216 of the microcard.

As shown in FIG. 6, the sample block platform may include a vacuum port260 positioned on the side 262 of the sample block platform. The vacuumport corresponds to the vacuum port that could be used in the FIGS. 1-4and FIG. 5 embodiments. The vacuum port 260 has an internal passageway264 that is fluidly connected to the channels 256 and 258 of the sampleblock platform. By connecting a vacuum device to the vacuum port, asubstantial vacuum can be drawn in a manner described below.

The thermal cycling device of FIGS. 6-8 may further include a microcardcarrier 270. As shown in FIGS. 6-8, the microcard carrier includes anumber of optical openings 272 that permit radiation to pass through thecarrier. Lenses may also be positioned in the optical openings, ifdesired. The microcard carrier may also include a downwardly projectingrib 274 for engagement with top surface 214 of microcard 212.

FIG. 8 illustrates a cross-sectional view of the apparatus of FIGS. 6-7,taken along line VIII-VIII in FIG. 6. The channels 256 in thisembodiment are not positioned immediately below the sample chambers 218.FIG. 8 shows a cross-section along a line passing through the samplechambers, therefore, the channel 256 is only shown as a dashed line. Asseen in FIG. 8, the spaces above the sample chambers 218 are surroundedby air, and are not in direct contact with the microcard carrier 270.The microcard may remain in firm contact with the upper surface 272 ofthe sample block platform 234 due to the operation of the vacuum device.

As is clear from the above descriptions of various embodiments, thepresent teachings include a method of maintaining a microcard on asample block of a thermal cycling device. The method can include thesteps of providing a sample block with a plurality of channels on anupper surface thereof. The method may further include the step ofproviding a space above the upper surface of the sample block for amicrocard containing at least one sample of biological material so thata bottom surface of the microcard may contact the upper surface of thesample block. A vacuum may then be imparted on the spaces defined by thechannels on the upper surface of the sample block and the bottom surfaceof the microcard positioned adjacent the upper surface of the sampleblock, the vacuum creating a force to urge the adjacent microcardagainst the upper surface of the sample block. The microcard may then bethermally cycled through a sequence of times and temperatures comprisingat least a first temperature maintained for a first period of time and asecond temperature maintained for a second period of time, with saidsecond temperature being higher than said first temperature.Simultaneously with the step of thermally cycling the microcard, theoptical characteristics of the at least one sample of biologicalmaterial or of one or more detectable markers associated therewith maybe detected. In accordance with various embodiments, a gasket may beprovided on an outer peripheral surface of the sample block, the gasketcontacting and forming a seal with the bottom surface of the microcardduring thermal cycling of the microcard.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure and methodsdescribed above. Thus, it should be understood that the presentteachings are not limited to the examples discussed in thespecification. Rather, the present teachings are intended to covermodifications and variations.

1. A thermal cycling device for thermally cycling samples of biologicalmaterial, the thermal cycling device comprising: a microcard having atop surface and a bottom surface, the top surface having a plurality ofchambers each containing samples of biological material and at least oneprimer; a sample block having an upper surface and a plurality ofchannels formed along the upper surface, the upper surface supportingthe bottom surface of the microcard such that the bottom surface of themicrocard overlays the plurality of channels to form a sealed volume; atemperature control system operably coupled to the sample block, thetemperature control system cycling the sample block through a polymerasechain reaction temperature cycle; and a vacuum device in fluidcommunication with the plurality of channels formed in the sample block,said vacuum device exerting and maintaining a vacuum within the sealedvolume to urge the microcard toward the sample block to maximize thermalcontact between the microcard and the sample block and permit generallyuniform and consistent temperature cycling of the microcard.
 2. Thethermal cycling device of claim 1 wherein the plurality of channelscomprise a plurality of perpendicularly intersecting grooves.
 3. Thethermal cycling device of claim 1 wherein the plurality of channelscomprise a plurality of substantially parallel grooves.
 4. The thermalcycling device of claim 1, further comprising: a gasket positionedaround an outer periphery of the plurality of channels in the sampleblock, the gasket configured to engage the bottom surface of themicrocard when the microcard is positioned on the sample block.
 5. Thethermal cycling device of claim 4, further comprising: an outer groovedisposed about the outer periphery of the channels in the sample block,the gasket being positioned in the outer groove.
 6. The thermal cyclingdevice of claim 1, further comprising: a microcard carrier positionedabove the microcard when the microcard is positioned on the sampleblock.
 7. The thermal cycling device of claim 6 wherein the microcardcarrier comprises: at least one downwardly projecting rib engaging anouter periphery of the top surface of the microcard, the rib pressingdownward on the outer periphery of the top surface of the microcard toassist in maintaining a vacuum within the sealed volume.
 8. The thermalcycling device of claim 7 wherein the microcard carrier comprises: atleast one opening generally aligned with at least one of the pluralityof chambers in the microcard.
 9. The thermal cycling device of claim 7,further comprising: a cover engaging and downwardly urging the microcardcarrier.